Method of attenuating reactions to skin irritants

ABSTRACT

The present invention is directed to a method of inhibiting CD1d activation by administering a composition containing a moiety that blocks CD1d activation. Compositions of the invention are useful for the attenuation of CD1d-restricted immune responses, including treatment of skin disorders due to hyperactive immune responses (e.g., contact hypersensitivity), for systemic administration to attenuate ongoing immune responses, and to provide hypoallergenic cosmetic products including pharmaceutical, cosmetic, and skin care compositions. Preferably, these compositions are in a form intended for topical administration.

CROSS-REFERENCE

This application is a Continuation Application of U.S. patentapplication Ser. No. 14/063,356, filed Oct. 25, 2013; which is aContinuation Application of U.S. patent application Ser. No. 12/146,386,filed Jun. 25, 2008; which is a Divisional Application of U.S. patentapplication Ser. No. 10/106,901, filed Mar. 26, 2002, now U.S. Pat. No.7,419,958; which claims the benefit of U.S. Provisional PatentApplication No. 60/278,837, filed Mar. 26, 2001, each of which isentirely incorporated herein by reference.

This invention was supported by National Institutes of Health Grants R01A145051, K11 DK02345, and R01 CA74886 and the government of the UnitedStates has certain rights thereto.

FIELD OF THE INVENTION

The present invention is directed to a method of attenuating and/orinhibiting a variety of reactions such as rashes caused by irritants.Preferably, the attenuated reaction is an immune response associatedwith CD1d activation.

BACKGROUND OF THE INVENTION

The skin represents the body's initial line of defense against theenvironment. The body is continually exposed to a barrage of molecules,many of which can cause an adverse reaction upon contact. Thesereactions can follow a spectrum from causing a dramatic and detrimentaleffect on an individual to skin discoloration and blotching.

The reactions include typical allergic dermatitis, including contactdermatitis and atopic dermatitis, as well as irritant dermatitis.

Contact dermatitis is an inflammation of the skin, which occurs when theskin comes in contact with substances that the skin is sensitive orallergic to. The reaction usually appears within 24-48 hours afterexposure to the allergen. Common symptoms include redness, itching andswelling. Sometimes blistering and weeping of the skin also develop. Theclinical symptoms of contact dermatitis can include acute eczemaaccompanied by erythema, edema, papula, vesicle, erosion, and itching.Repeated exposure to an irritant can lead to the development of eczemaaccompanying lichenification and infiltration. Allergic contactdermatitis can appear after initial or prolonged exposure to anirritant. Contact dermatitis includes irritant dermatitis, phototoxicdermatitis, allergic dermatitis, photoallergic dermatitis, contacturticaria, systemic contact-type dermatitis and the like.

A wide range of agents can cause allergic contact dermatitis includingfor example, metals (e.g. nickel, chromium, cobalt), fragrances,chemicals, cosmetics, textiles, pesticides, plastics, and pollen (see,for example, R. J. G. Rycroft et al. “Textbook of Contact Dermatitis”).

Therapeutic agents such as drugs may also cause allergic contactdermatitis, particularly when administered transdermally. It is wellknown that many drugs, e.g., topical ointments, including some currentlymarketed in the United States (e.g. clonidine) sensitize the skin whenused.

Skin sensitization may be produced not only by transdermally delivereddrugs, but also by a non-sensitizing drug combined with skin sensitizingpermeation enhancers, or a combination of a sensitizing drug and asensitizing permeation enhancer. Penetration of these sensitizing agentsinto the skin and the resulting adverse reaction of the skin may persistwell beyond the time that the transdermal patch is removed from theskin. The reaction of the skin may be a source of discomfort and aclinical complication in patients suffering from such a reaction.

Atopic dermatitis is developed by exposure to various antigens, since anindividual has an atopic disposition which is hypersensitivity against acertain substance. The clinical symptoms include marked itching, skinhypertrophy, infiltration, lichenification and the like.

Irritant dermatitis can occur when too much of a substance is used onthe skin or when the skin is sensitive to a certain substance.Susceptibility can include a genetic component. Skin-irritating agentsare substances (e.g. soap) that cause an immediate and generallylocalized adverse response. The response is typically in the form ofredness and/or inflammation and generally does not extend beyond theimmediate area of contact. Symptoms that are commonly seen includeredness, scaling, and the skin looking irritated and sore.

Psoriasis is a skin condition associated with hyper-proliferation ofskin cells and immunologic involvement. Psoriasis is a common,idiopathic chronic skin disease characterized by inflamed, scaling, skinlesions containing infiltrates of neutrophils, lymphocytes, andmonocytes. Psoriasis manifests in many forms, including cutaneous,mucosal, ungual, and even psoriatic rheumatism. The most effectivetreatment in the control of localized psoriasis for most patients is theuse of topical corticosteroids and topical coal-tar preparations. Withcertain patients who have generalized psoriasis, it has been necessaryto use a variety of systemic chemotherapeutic agents, especiallymethotrexate.

Certain irritants may cause both allergic and non-allergic contactdermatitis. For example, latex. Latex refers to a type of plastic madefrom the milky sap of the rubber tree, and contains many proteins whichcan cause allergic reactions in sensitive individuals. Symptoms canrange from watery eyes, hives, rash, swelling, wheezing and in severecases, anaphylaxis. These responses can occur when latex items touch theskin; the mucous membranes (including the mouth, bladder, genitals, orrectum), and open wounds or bloodstream (especially during surgery).Anybody can develop latex sensitivity. People at increased risk fordeveloping latex allergy include workers with ongoing latex contact(like health care workers), persons with many environmental allergies(hay fever), and those with spina bifida. Latex is found in a wide arrayof common products, including, for example: gloves, balloons, band-aids,tourniquets, bandages, catheters, rubber bands, IV, other tubing (ex.stethoscopes), art supplies, pacifiers, bottle nipples, diapers,condoms/diaphragms, elastic, chewing gum, carpeting, hand grips ofbicycles and motorcycles, shoe soles, auto tires, swimming goggles andequipment.

The more common reaction to latex products is not allergic, but rather,irritant contact dermatitis, which can cause dry, itchy, irritated areason the skin, usually the hands. Skin reactions include a rash thatusually begins 24 to 48 hours after contact. It may progress to oozingblisters or spread away from area touched by latex. Latex allergy(immediate hypersensitivity) is a more serious reaction. Certainproteins in latex cause an allergic reaction. The amount of exposureneeded to cause symptoms is not known. Very low levels of exposure cantrigger allergic reactions in some people, while having no affect tomost people. Reactions usually begin within minutes of exposure tolatex, but can occur hours later and have a variety of symptoms. Mildreactions to latex usually cause skin redness, hives, or itching. Moresevere reactions can cause respiratory or breathing symptoms such asrunny nose, sneezing, itchy eyes, scratchy throat, and asthma (troublebreathing, coughing, and wheezing).

Individuals can also develop allergic dermatitis and/or irritantdermatitis in response to insects and plants and shrubbery. For example,certain plants such as poison ivy excrete chemicals that upon contactcan cause adverse reactions in humans. These reactions may particularlyoccur during gardening or nature walks.

Many animals can also suffer from a variety of skin irritations andinflammations generally known as dermatitis. For example, all animalscan develop contact dermatitis caused by flea, mosquito, or other insectbites, allergies, external stimulation such as from prickly plants, andfor other reasons. The condition has been notoriously difficult totreat. Veterinarians occasionally resort to injections of variousmedicines in an attempt to alleviate the symptoms and cure thedermatitis.

Presently known therapies for treating or preventing reactionsassociated with such irritants are inadequate. For example, steroidalagents and antihistamine agents have been used as therapeutic agents forcontact dermatitis, and these and a part of the so-called anti-allergicagents have been used for atopic dermatitis. The most widely prescribeddrugs to treat dermatologic disease are corticosteroids, also known asglucocorticosteroids or glucocorticoids. Approximately 50% ofprescriptions written by dermatologists are for topical corticosteroids.However, these drugs can cause adverse reactions and/or are not fullyeffective. Systemic corticosteroids are often required in some severedermatologic diseases but topical treatment is preferred in mostresponsive cases because it causes fewer systemic adverse effects.

Individual topical corticosteroid preparations vary in anti-inflammatorypotency and clinical efficacy.

Though some steroids, particularly mid- to high-potency steroids, areefficacious in chronic dermatoses, long term use of steroids isassociated with serious local side effects. These include skin atrophy(thinning, telangiectasia, striae) and a prompt rebound flare when thesteroid is stopped. Treatment of large areas of skin and use ofocclusive dressings can also increase the potential for adverse effects.This is especially the case in children.

Examples of anti-histamine agents include diphenhydramine hydrochloride,mequitazine, promethazine hydrochloride, and chlorpheniramine maleateanti-histamines have been used mainly to reduce itchiness. Anti-allergicagents include tranilast, ketotifen fumarate, oxatomide, and azelastinehydrochloride. In general, conventional so-called antiallergic agentsare either ineffective or fail to show satisfactory therapeutic effectson contact dermatitis and atopic dermatitis.

Accordingly, there is a need for a treatment for skin reactionsincluding allergic dermatitis (contact dermatitis and atopicdermatitis), as well as irritant dermatitis.

The pathophysiologic mechanisms involved in the above-described skindisorders and the evolution of such inflammatory processes are poorlyunderstood. There are numerous skin conditions characterized byincreased T cell activation and abnormal antigen presentation in thedermis and epidermis. Thus, there has been speculation that skin cellsare important in the generation of a cutaneous inflammatory response(Kupper, “Immune and Inflammatory Processes in Cutaneous Tissues”, J.Clin. Invest., 86, pp. 1783-89 (1990)).

CD1d-restricted NK T cells are among the immune system cells found inthe skin. The in vivo functions of CD1d-restricted NK T cells are notfully known. They are involved in the IgG response to GPI-anchoredproteins of various parasites, contribute to the IL-12-mediatedrejection of tumors, and appear to regulate some autoimmune disordersand clearance of certain infections through the production of cytokines.Many of these functions have been observed by using lipid ligands thatbind CD1d and activate NK T cells. For example, α-gal-cer.

In humans, the direct cellular targets for their immunomodulatoryfunction(s) have remained enigmatic.

SUMMARY OF THE INVENTION

We have now discovered that one can attenuate and block CD1d activation.This can be done by administering compositions that attenuateCD1d-restricted NK T cell responses. Such compositions include compoundsthat act as antagonists by binding CD1d and inhibiting activation ofCD1d-restricted NK T cells, compounds that block CD1d-specific receptorson NK T cells, and decoys, mimics and the like. Compositions includepharmaceutical and cosmetic compositions.

One preferred group of compositions include phospholipids that bind CD1dwithout activating NK T cells. Preferred phospholipids include1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine (DPPE) and1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-[Poly(ethyleneglycol) 2000](DPPE-PEG).

Another preferred group of compositions includes glycolipid antagoniststhat bind CD1d without activating NK T cells. Preferred glycolipidsinclude ceramide, αMan Cer, and βGal Cer.

Another preferred group of compositions includes phosphatidyl inositol.

This method of attenuating CD1d activation can be used for treatment ofconditions associated with activation of CD1d-restricted NK T cells.Preferred conditions include skin disorders due to hyperactiveCD1d-restricted T cell responses and other disorders associated withongoing CD1d-restricted immune responses.

Such CD1d associated skin disorders include contact dermatitis andfurther eczematous dermatitises, atopical dermatitis, seborrheicdermatitis, psoriasis, Lichen planus, Pemphigus, bullous Pemphigoid,epidermolysis bullosa, urticaria, angioedemas, vasculitides, erythemas,cutaneous eosinphilias, Lupus erythematosus, and Alopecia areata.

A preferred embodiment of the invention is directed to the treatment ofcontact hypersensitivity.

Another embodiment of the invention provides hypoallergenic cosmeticproducts.

Another embodiment of the invention is directed to compositions that canbe used prophylactically to prevent a reaction before one encounters theirritant. For example, a composition to use before weeding.

One preferred embodiment of the invention provides preventing ortreating skin sensitization produced by topical administration oftherapeutic drugs.

Other embodiments of the invention provide systemic administration ofcompositions to attenuate ongoing CD1d-restricted immune responses.Systemic therapy can be used in any individual for which activation ofNKT cells would be adverse. Systemic conditions can include individualswith autoimmune disease such as lupus. It can also be used in women athigh risk for spontaneously aborting pregnancies.

In one embodiment of the invention, a locally administrable topicalpharmaceutical or cosmetic composition is provided for the attenuationor treatment of skin conditions associated with CD1d-restricted immuneresponses. The locally administrable topical pharmaceutical compositionincludes a topical carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing that contact hypersensitivity is ablated inCd1d knock out mice and in iNKT cell knock out mice. Mice at least sevenweeks of age were sensitized epicutaneously on day 0 with 150 μl of 3%oxazolone in 100% ethanol on the shaved abdomen. 6 days later the rightear was challenged with 1% oxazolone, 10 μl on each side or carrieronly. The hapten specific increase in ear thickness at 24 hours wasdetermined using a micrometer.

FIG. 2 is a graph showing that systemic treatment with α-ManCerinhibited oxazolone-induced contact hypersensitivity. Mice at leastseven weeks of age were sensitized epicutaneously on day 0 with 70 μl of4% oxazolone in acetone/olive oil (4/1) with or without treatment withα-GalCer or α-ManCer. The hapten specific increase in ear thickness at24 hours was determined using a micrometer. 5 days later the right earwas challenged with 0.5% oxazolone, 10 μl on each side or carrier only.The hapten specific increase in ear thickness at 24 hours was determinedusing a micrometer.

FIG. 3 is a graph showing that topical DPPE-PEG (a CD1d-binding lipid)inhibits oxazolone-induced contact hypersensitivity. Mice at least sevenweeks of age were sensitized epicutaneously on day 0 with 70 l of 4%4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (oxazolone, Sigma) inacetone/olive oil (4/1) with or without DPPE-PEG (100 mg/ml) andchallenged five days later on the ear with 20 l of 0.5% oxazolone orcarrier only. The hapten-specific increase in ear thickness at 24 hourswas determined with a micrometer.

FIG. 4A is a dose response curve showing that topical administration ofDPPE-PEG inhibits oxazolone-induced contact hypersensitivity in a dosedependent fashion. Mice at least seven weeks of age were sensitizedepicutaneously on day 0 with 70 l of 4% oxazolone in acetone/olive oil(4/1) with DPPE-PEG in a series of increasing dosage, ranging from 0 to100 mg/ml. 5 days later the right ear was challenged with 0.5%oxazolone, 10 l on each side or carrier only. The hapten specificincrease in ear thickness at 24 hours was determined using a micrometer.FIG. 4B is a graph showing topical administration of DPPE-PEG inhibitsoxazolone-induced contact hypersensitivity both during recall andpriming. Mice at least seven weeks of age were sensitized epicutaneouslyon day 0 with 70 l of 4% oxazolone in acetone/olive oil (4/1) with orwithout DPPE-PEG. 5 days later the right ear was challenged with 0.5%oxazolone, 10 l on each side or carrier only. One group that was primedwith oxazolone only was challenged with a mixture of oxazolone 0.05% andDPPE-PEG (100 mg/ml). The hapten specific increase in ear thickness at24 hours was determined using a micrometer.

FIG. 5 is a graph showing that inhibition of CSH by DPPE-PEG wasindependent of MHC alleles. In bred strains of mice with unrelated MHCalleles were challenged with oxazolone with or without DPPE-PEG at 100mg/ml as described for FIG. 4. The application of antagonist lipidinhibited CHS responses in SWR, C57B1/10, DBA, and BALB/c mice.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method of inhibiting CD1dactivation. The method comprises administering a composition containinga moiety that blocks CD1d activation.

Compositions of the invention are useful for the attenuation ofCD1d-restricted immune responses, including treatment of skin disordersdue to hyperactive immune responses (e.g., contact hypersensitivity),for systemic administration to attenuate ongoing immune responses, andto provide hypoallergenic cosmetic products including pharmaceutical,cosmetic, and skin care compositions. Preferably, these compositions arein a form intended for topical administration.

We have discovered that certain antagonists bind CD1d and inhibitactivation of NK T cells. Consequently, by exposing CD1d-expressingcells to a CD1d-specific antagonist, one can attenuate immune responsesby specific inhibition of CD1d-restricted events. Antagonists of thepresent invention include any molecules that inhibit activation ofCD1d-restricted NK T cells. Thus, one can treat subjects suffering froman activated CD1d associated disorder such as a skin disorder byadministering an effective amount of CD1d specific antagonist.

Such compounds include antagonists that bind CD1d and inhibit NK T cellactivation, antagonists that block CD1d-specific receptors such as on NKT cells, decoys that prevent CD1d binding to the CD1d specific receptor,and the like.

Experiments in human models have revealed that CD1d-restricted CD161+ Tcells specifically target myeloid dendritic (DC1) cells. DC1 dendriticcells are integral to the genesis of Th1 immune responses. Accordingly,their susceptibility to lysis by CD1d-restricted CD161+ T cells may bepart of a negative feedback loop in cell-mediated immune responses.

We tested dendritic cell function in CD1d null and wild type mice. TheCD1d mull mice had impaired priming responses in mixed lymphocytereactions, to tumor vaccinations, and in skin contact hypersensitivity(see FIG. 1). In addition, contact hypersensitivity could be blocked bythe cutaneous addition of lipid ligands know to bind CD1d but notactivate NK T cells (see FIG. 2).

Accordingly, one can readily determine whether a compound inhibitsactivation of CD1d by looking at activation of CD1d-restricted NK Tcells in vitro using standard assays such as described herein. Forexample, the proliferation of NK T cells is indicative of theiractivation by binding to CD1d-expressing cells. Proliferation can bemeasured, for example, by determining the incorporation of [³H]thymidineinto Vα14 NK T cells (Kawano et al., Science 278:1626-29 (1997)). Otherin vitro assays include the induction of cytokine production.

CD1d-specific antagonists include any antagonist that binds CD1d andinhibits activation of NK T cells. The binding of an antagonist to CD1dcan be determined in vitro using standard assays. For example, surfaceplasmon resonance (Naidenko et al., J. Exp. Med. 190:1069-79 (1999)).Molecules known to bind CD1d include antibodies, phospholipids andglycolipids, including highly glycosylated sphingolipids (gangliosides)(Kawano et al., Science 278: 1626-9 (1997); Naidenko et al., J. Exp.Med. 190:1069-79 (1999); Briken et al., Sem. Immunol. 12: 517-25 (2000);Kronenberg et al., Proc. Natl. Acad. Sci. USA 98: 2950-52 (2001)).

Phospholipids that bind CD1d without activating NK T cells include1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine (DPPE) (Naidenko etal., 1999). The phospholipid may be coupled to a conjugate, such asother lipids coupled to a carrier such as biotin or a poly(alkalineoxide), for example polyethylene glycol (PEG). Polymeric substances suchas dextran, polyvinyl pyrrolidones, polysaccharides, starches, polyvinylalcohols, polyacryl amides or other similar polymers can be used.Polyethylene glycol (PEG) as the poly(alkylene oxide) is preferred. Thepoly(alkylene oxides) can include monomethoxy polyethylene glycol,polypropylene glycol, block copolymers of polyethylene glycol andpolypropylene glycol and the like. The polymers can also be distallycapped with C₁₋₄ alkyls instead of monomethoxy groups.

Other preferred phospholipids include phosphatidyl inositol.

Glycolipids that bind to CD1d share a common motif consisting of ahydrophobic portion composed of a branched or dual alkyl chain moietywith a covalently linked hydrophilic cap formed by a polar or chargedgroup of the lipid of associated carbohydrates (Briken et al., 2000).The prototypical glycolipid antigen presented by CD1d is-galactosylceramide (Kawano et al., 1997). Thus, D1d antagonists caninclude glycolipids such as monoglycosylated ceramides anddiglycosylated ceramides (Kawano et al., 1997).

Examples of monoglycosylated ceramides and diglycosylated ceramideswhich bind CD1d but do not activate NK T cells can include ceramideswith inner sugar groups at the β-anomer position (such asGalα1-4Glcβ1-1′Cer), an axial configuration of the 2-hydroxyl group(such as α-ManCer), derivatives lacking the 3- and 4-hydroxyl groups onthe phytosphingosine of α-GalCer (such as 3,4-deoxy α-GalCer), andceramides with fatty acyl chain with less than C₂₆, and a sphingosinebase less than C₁₈. Glycolipid antagonists can also be coupled to aconjugate such as biotin or a poly(alkylene oxide), for example PEG.

Other CD1d glycolipid antagonists include highly glycosylatedsphingolipids, also known as gangliosides. Ganglioside antagonistsinclude GM1 and GD1a (Naidenko et al., 1997). The antagonists can becoupled to a conjugate such as a biotin or poly(alkylene oxide).

Preferred examples of CD1d-specific lipid antagonists include but arenot limited to: DPPE-PEG, phosphatidyl inositol, ceramide, α-ManCer,β-GalCer, Galα1-4Glcβ1-1′Cer, and 13,4-deoxy α-GalCer, GM1, and GD1a.

Other antagonists can include antibodies that specifically bind to CD1dand in doing so prevent CD1d from binding to a CD1d specific receptor.Single chain antibodies and humanized monoclonal antibodies arepreferred. Alternatively, one can use molecules that block theCD1d-specific receptor. For example, a molecule. Alternatively, one canmodify the lipids that activate CD1d binding by capping the end thatbind to CD1d (Kawano et al., Science 278: 1626-9 (1997); Naidenko etal., J. Exp. Med. 190:1069-79 (1999); Briken et al., Sem. Immunol. 12:517-25 (2000); Kronenberg et al., Proc. Natl. Acad. Sci. USA 98: 2950-52(2001)).

One can also use decoys that mimic CD1d receptors so that the CD1dmolecule does not bind to the receptor.

CD1d blocking molecules are presently preferred.

Compositions of the invention should be physiologically stable attherapeutically effective concentrations. Physiological stable compoundsare compounds that do not break down or otherwise become ineffectiveupon introduction to a patient prior to having a desired effect.Compounds are structurally resistant to catabolism, and thus,physiologically stable, or coupled by electrostatic or covalent bonds tospecific reagents to increase physiological stability. Such reagentsinclude amino acids such as arginine, glycine, alanine, asparagine,glutamine, histidine or lysine, nucleic acids including nucleosides ornucleotides, or substituents such as carbohydrates, saccharides andpolysaccharides, lipids, fatty acids, proteins, or protein fragments.Useful coupling partners include, for example, glycol such aspolyethylene glycol, glucose, glycerol, glycerin and other relatedsubstances.

Preferably, the compositions are not substantially toxic, myelotoxic,mutagenic or teratogenic at required dosages. Although side effects mayoccur, preferably the benefits achieved from their use outweighdisadvantages attributable to adverse side effects.

Compositions of the present invention can be used to prevent or treatany condition associated with activation of CD1d-restricted NK T cells,in which attenuation of CD1d activation, such as attenuation of theCD1d-restricted NK T cell response, would be desirable. Preferredembodiments of the invention include topical administration to treatskin disorders due to hyperactive immune responses (e.g., contacthypersensitivity) and systemic administration. Prophylactic use toattenuate immune responses. For example, to avoid reactions to certainplants such as poison ivy, poison oak, etc. when you are going to be inan area where exposure to such a substance is likely, for example, ifyou are going to be in the woods, gardening, etc.

Systemic administration is preferred in any individual for whichactivation of NKT cells would be adverse.

Certain women have a problem with spontaneous abortion which appears tobe associated with high levels of CD1d. These individuals could use thepresent compositions before trying to conceive, preferably usingsystemic administration.

Certain individuals with autoimmune diseases have complicationsassociated in part with CD1d activation. These compositions can be usedwith such individuals.

One problem with allergic or irritant caused itching is that thescratching can in fact result in further irritation that causes rashesand irritation long after the initial stimulus is gone. This is aparticular problem with non-human animals. These compositions can beused to attenuate, treat or present such conditions. Another preferredembodiment includes the composition in hypoallergenic cosmetic products.

In one embodiment of the invention, a locally administrable topicalpharmaceutical composition is provided for the prevention or treatmentof skin conditions associated with CD1d-restricted T cell responses.Skin conditions include, but are not limited to, contact dermatitis andfurther eczematous dermatitises, atopical dermatitis, seborrheicdermatitis, Lichen planus, Pemphigus, bullous Pemphigoid, epidermolysisbullosa, urticaria, angioedemas, vasculitides, erythemas, cutaneouseosinphilias, Lupus erythematosus, and Alopecia areata.

A preferred skin condition is contact dermatitis. Contact dermatitis canbe caused by a variety of irritants. The most widely known naturalallergens which are capable of sensitizing and causing contactdermatitis in many people are antigenic plants of the genus Rhus, suchas poison ivy, poison oak, and poison sumac. Other widely known skinirritants are commercial products such as insecticides containingPyrethrum or Rotenone, dye intermediates such as aniline, nitrocompounds, anthracene, and derivatives thereof, dyes such asparaphenylenediamine and aniline black, photo developers such ashydroquinone and para-amido-phenol, antioxidants such as hexamethylenetetramine, and synthetic and natural resins such as wood rosin andphenol formaldehyde, and detergents and constituents of rubber and latexgloves

Other agents that cause contact dermatitis include, for example, metals(e.g. nickel, chromium, cobalt, including exposure during bodypiercing), latex, fragrances, chemicals, cosmetics, textiles,pesticides, plastics, pollen, and the like. Therapeutic agents such asdrugs may also cause allergic contact dermatitis, particularly whenadministered topically or transdermally. Agents that cause non-allergiccontact dermatitis include skin-irritating agents such as water, skincleansers, industrial cleaning agents, alkalis, acids, oils, organicsolvents, oxidizing agents, reducing agents, plant matter, animalmatter, combinations thereof, and the like.

Another preferred skin condition is psoriasis. Psoriasis is a skincondition associated with hyper-proliferation of skin cells andimmunologic involvement. Psoriasis is a common, idiopathic chronic skindisease characterized by inflamed, scaling, skin lesions containinginfiltrates of neutrophils, lymphocytes, and monocytes. The compositionsand methods of the present invention can be used to treat any form ofpsoriasis, including cutaneous, mucosal, ungual, and even psoriaticrheumatism. Compositions and methods of the present invention can beused to treat localized and generalized psoriasis.

One preferred embodiment of the invention provides prophylactictreatment to minimize skin sensitization produced by topicaladministration of therapeutic drugs. In another embodiment, thecomposition can be administered with the therapeutic drug or cosmetic.In still another embodiment, the composition is used for treatment afterthe reaction has occurred.

Another embodiment of the invention provides systemic administration ofcompositions to attenuate ongoing CD1d-restricted immune responses. Thisembodiment is preferable for any individual for whom activation of NKTcells would be adverse. Systemic conditions can include certain highrisk spontaneously aborting pregnancies. It can also be used withautoimmune diseases such as lupus.

In another embodiment of the invention, a locally administrable topicalcosmetic composition is provided, for example to provide hypoallergenicproducts.

The compositions of the present invention include those suitable fortopical and systemic administration including oral, rectal,intravaginal, nasal, ophthalmic or parenteral administration, all ofwhich may be used as routes of administration using the materials of thepresent invention. A preferred route of administration is topical. Thetopical composition may be in the form of a pharmaceutical but it doesnot have to be. For example, it can be a cosmetic.

In one embodiment of the invention, the locally administrable topicalcomposition is provided for the prevention or treatment of skinconditions associated with CD1d-restricted immune responses. The locallyadministrable topical composition includes a topical carrier.

The topical carrier, as noted above, is one which is generally suited totopical drug administration and includes any such materials known in theart. The topical carrier is selected so as to provide the composition inthe desired form, e.g., as a liquid, lotion, cream, paste, gel, powder,or ointment, and may be comprised of a material of either naturallyoccurring or synthetic origin. It is essential that the selected carriernot adversely affect the active agent or other components of the topicalformulation. Examples of suitable topical carriers for use hereininclude water, alcohols and other nontoxic organic solvents, glycerin,mineral oil, silicone, petroleum jelly, lanolin, fatty acids, vegetableoils, parabens, waxes, and the like. The composition of the inventionmay also be administered in the form of a shampoo, in which caseconventional components of such a formulation are included as well,e.g., surfactants, conditioners, viscosity modifying agents, humectants,and the like.

Particularly preferred formulations herein are colorless, odorlessointments, lotions, creams and gels.

Ointments are semisolid preparations which are typically based onpetrolatum or other petroleum derivatives. The specific ointment base tobe used, as will be appreciated by those skilled in the art, is one thatwill provide for optimum drug delivery, and, preferably, will providefor other desired characteristics as well, e.g., emolliency. As withother carriers or vehicles, an ointment base should be inert, stable,nonirritating and nonsensitizing. As explained in Remington: The Scienceand Practice of Pharmacy, 19th Ed. (Easton, Pa.: Mack Publishing Co.,1995), at pages 1399-1404, ointment bases may be grouped in fourclasses: oleaginous bases; emulsifiable bases; emulsion bases; andwater-soluble bases. Oleaginous ointment bases include, for example,vegetable oils, fats obtained from animals, and semisolid hydrocarbonsobtained from petroleum. Emulsifiable ointment bases, also known asabsorbent ointment bases, contain little or no water and include, forexample, hydroxystearin sulfate, anhydrous lanolin and hydrophilicpetrolatum. Emulsion ointment bases are either water-in-oil (W/O)emulsions or oil-in-water (OW) emulsions, and include, for example,cetyl alcohol, glyceryl monostearate, lanolin and stearic acid.Preferred water-soluble ointment bases are prepared from polyethyleneglycols of varying molecular weight (Remington: The Science and Practiceof Pharmacy).

Lotions are preparations to be applied to the skin surface withoutfriction, and are typically liquid or semiliquid preparations in whichsolid particles, including the active agent, are present in a water oralcohol base. Lotions are usually suspensions of solids, and preferably,for the present purpose, comprise a liquid oily emulsion of theoil-in-water type. Lotions are preferred formulations herein fortreating large body areas, because of the ease of applying a more fluidcomposition. It is generally necessary that the insoluble matter in alotion be finely divided. Lotions will typically contain suspendingagents to produce better dispersions as well as compounds useful forlocalizing and holding the active agent in contact with the skin, e.g.,methylcellulose or sodium carboxymethyl-cellulose, or the like. Aparticularly preferred lotion formulation for use in conjunction withthe present invention contains propylene glycol mixed with a hydrophilicpetrolatum such as that which may be obtained under the trademarkAquaphor® from Beiersdorf, Inc. (Norwalk, Conn.).

Creams containing the selected agent are, as known in the art, viscousliquid or semisolid emulsions, either oil-in-water or water-in-oil.Cream bases are water-washable, and contain an oil phase, an emulsifierand an aqueous phase. The oil phase, also sometimes called the“internal” phase, is generally comprised of petrolatum and a fattyalcohol such as cetyl or stearyl alcohol; the aqueous phase usually,although not necessarily, exceeds the oil phase in volume, and generallycontains a humectant. The emulsifier in a cream formulation, asexplained in Remington, supra, is generally a nonionic, anionic,cationic or amphoteric surfactant.

Gels formulations are preferred for application to the scalp. As will beappreciated by those working in the field of topical drug formulation,gels are semisolid, suspension-type systems. Single-phase gels containorganic macromolecules distributed substantially uniformly throughoutthe carrier liquid, which is typically aqueous, but also, preferably,contain an alcohol and, optionally, an oil.

Shampoos may be formulated with the standard shampoo components, i.e.,cleansing agents, thickening agents, and preservatives with thecleansing agent representing the primary ingredient, typically ananionic surfactant or a mixture of an anionic and an amphotericsurfactant.

Various additives, known to those skilled in the art, may be included inthe topical formulations of the invention. For example, solvents may beused to solubilize certain drug substances. Other optional additivesinclude skin permeation enhancers, opacifiers, anti-oxidants, gellingagents, thickening agents, stabilizers, and the like. Other agents mayalso be added, such as antimicrobial agents, antifungal agents,antibiotics and anti-inflammatory agents such as steroids.

In the preferred topical formulations of the invention, the active agentis present in an amount which is generally less than 10% by weight ofthe total composition, preferably less than about 1% by weight, and mostpreferably less than about 0.1% by weight.

The topical compositions of the invention may also be delivered to theskin using a time-release mechanism. For example, “transdermal”-typepatches, wherein the CD1d activation-blocking composition is containedwithin a laminated structure that serves as a drug delivery device to beaffixed to the skin. In such a structure, the drug composition iscontained in a layer, or “reservoir,” underlying an upper backing layer.The laminated structure may contain a single reservoir, or it maycontain multiple reservoirs. In one embodiment, the reservoir comprisesa polymeric matrix of a pharmaceutically acceptable contact adhesivematerial that serves to affix the system to the skin during drugdelivery. Examples of suitable skin contact adhesive materials include,but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes,polyacrylates, polyurethanes, and the like. The particular polymericadhesive selected will depend on the particular drug, vehicle, etc.,i.e., the adhesive must be compatible with all components of thedrug-containing composition. In an alternative embodiment, thedrug-containing reservoir and skin contact adhesive are present asseparate and distinct layers, with the adhesive underlying the reservoirwhich, in this case, may be either a polymeric matrix as describedabove, or it may be a liquid or hydrogel reservoir, or may take someother form.

The backing layer in these laminates, which serves as the upper surfaceof the device, functions as the primary structural element of thelaminated structure and provides the device with much of itsflexibility. The material selected for the backing material should beselected so that it is substantially impermeable to the CD1dactivation-blocking composition and to any other components of thecomposition, thus preventing loss of any components through the uppersurface of the device. The backing layer may be either occlusive ornonocclusive, depending on whether it is desired that the skin becomehydrated during drug delivery. The backing is preferably made of a sheetor film of a preferably flexible elastomeric material. Examples ofpolymers that are suitable for the backing layer include polyethylene,polypropylene, polyesters, and the like.

During storage and prior to use, the laminated structure preferablyincludes a release liner. Immediately prior to use, this layer isremoved from the device to expose the basal surface thereof, either thedrug reservoir or a separate contact adhesive layer, so that the systemmay be affixed to the skin. The release liner should be made from adrug/vehicle impermeable material.

Such devices may be fabricated using conventional techniques, known inthe art, for example by casting a fluid admixture of adhesive, drug andvehicle onto the backing layer, followed by lamination of the releaseliner. Similarly, the adhesive mixture may be cast onto the releaseliner, followed by lamination of the backing layer. Alternatively, thedrug reservoir may be prepared in the absence of drug or excipient, andthen loaded by “soaking” in a drug/vehicle mixture.

As with the topical formulations of the invention, CD1d activationblocking composition contained within the reservoirs of these laminatedsystem may contain a number of components. In some cases, the blockingcomposition may be delivered “neat.” i.e., in the absence of additionalliquid. In most cases, however, the composition will be dissolved,dispersed or suspended in a suitable pharmaceutically acceptablevehicle, typically a solvent or gel. Other components which may bepresent include preservatives, stabilizers, surfactants, and the like.

Preferably, the topical formulations and the laminated delivery systemsalso contain a skin permeation enhancer. A skin permeation enhancer canbe co-administered. Suitable enhancers are well know in the art andinclude, for example, dimethylsulfoxide (DMSO), dimethyl formamide(DMF), N,N-dimethylacetamide (DMA), decylmethylsulfoxide (C₁₀ MSO),C₂-C₆ alkanediols, and the 1-substituted azacycloheptan-2-ones,particularly 1-n-dodecylcyclazacycloheptan-2-one (available under thetrademark Azone® from Whitby Research Incorporated, Richmond, Va.),alcohols, and the like.

The ointments, pastes, creams and gels also may contain excipients, suchas animal and vegetable fats, oils, waxes, paraffins, starch,tragacanth, cellulose derivatives, polyethylene glycols, silicones,bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.Powders and sprays also can contain excipients such as lactose, talc,silicic acid, aluminum hydroxide, calcium silicates and polyamidepowder, or mixtures of these substances. Sprays can additionally containcustomary propellants, such as chlorofluorohydrocarbons and volatileunsubstituted hydrocarbons, such as butane and propane.

The topical compositions according to this invention may also includeone or more preservatives or bacteriostatic agents, e.g., methylhydroxybenzoate, propyl hydroxybenzoate, chlorocresol, benzalkoniumchlorides, and the like. The topical compositions also can contain otheractive ingredients such as antimicrobial agents, particularlyantibiotics, anesthetics, analgesics, and antipruritic agents.

Antimicrobial agents that can be used in the topical administration ofthe present invention are those compatible with skin and soluble insolvents. In addition, antimicrobial agents are active against a broadspectrum of microorganisms, including but are not limited to, grampositive and gram negative bacteria, yeast, and mold. Examples of theantimicrobial agents include, but are not limited to, triclosan(5-chloro-2-(2,4-dichlorophenoxy) phenol which is also known as Irgasan™DP 300 manufactured by Ciba-Geigy Corporation), hexetidine(5-amino-1,3-bis(2-ethylhexyl)-5-methyl-hexahydropyrimidine),chlorhexidine salts (salts ofN,N″-Bis(4-chlorophenyl)-3,12-diimino-2,4,11,14-tetraazatetradecanediimidiamide),2-bromo-2-nitropropane-1,3-diol, hexyresorcinol, benzalkonium chloride,cetylpyridinium chloride, alkylbenzyldimethylammonium chlorides, iodine,phenol derivatives, povidone-iodine (polyvinylpyrrolidinone-iodine),parabens, hydantoins (2,4-imidazolidinedione), hydantoins derivatives(derivatives of 2,4-imidazolidinedione), phenoxyethanol, cis isomer of1-(3-chloroallyl)-3,5,6-triaza-1-azoniaadamantane chloride(quarternium-15 which is also known as Dowicil 200 manufactured by DowChemical Company), diazolidinyl urea, benzethonium chloride,methylbenzethonium chloride, and mixtures thereof. Examples ofhyndantoin derivatives include, but are not limited to,dimethylol-5,5-dimethylhydantoin (glydant). Preferably, examples of theantimicrobial agents include triclosan, cis isomer of1-(3-chloroallyl)-3,5,6-triaza-1-azoniaadamantane chloride(quarternnium-15), hyndantoins, hyndantoin derivatives such asdimethylol-5,5-dimethylhydantoin (glydant), and mixtures thereof.

The percentage of the antimicrobial agents in the composition is about0.01 wt. % to about 10 wt. %, and preferably, about 0.01 wt. % to about2 wt. %.

In one embodiment such as where a latex condom or diaphragm is to beused, the CD1d activation block compositions can be included inointments, foams, and creams that can be used during sex. For example,they can be administered preferably prior to or just after sexualcontact such as intercourse. One preferred composition would be avaginal foam, including a spermicide, containing one of the compounds.

The topical compositions and drug delivery systems of the invention canbe used in the prevention or treatment of the skin conditions identifiedabove. When used in a preventive (prophylactic) method, susceptible skincan be treated prior to exposure or just after exposure but any visiblelesions on areas known to be susceptible to such lesions are observed.In treating skin conditions, it will be recognized by those skilled inthe art that the optimal quantity and spacing of individual dosages willbe determined by the nature and extent of the condition being treated,the form, route and site of administration, and the particularindividual undergoing treatment, and that such optimums can bedetermined by conventional techniques. It will also be appreciated byone skilled in the art that the optimal dosing regimen, i.e., the numberof doses can be ascertained using conventional course of treatmentdetermination tests. Generally, a dosing regimen will involveadministration of the selected topical formulation at least once daily,and preferably one to four times daily, until the symptoms havesubsided.

Systemic administration of a composition may be by oral, parenteral,sublingual, rectal such as suppository or enteral administration, or bypulmonary absorption. Parenteral administration may be by intravenousinjection, subcutaneous injection, intramuscular injection,intra-arterial injection, intrathecal injection, intra peritonealinjection or direct injection or other administration to one or morespecific sites. When long term administration by injection is necessary,venous access devices such as medi-ports, in-dwelling catheters, orautomatic pumping mechanisms are also preferred wherein direct andimmediate access is provided to the arteries in and around the heart andother major organs and organ systems.

Compositions may also be administered to the nasal passages as a spray.Arteries of the nasal area provide a rapid and efficient access to thebloodstream and immediate access to the pulmonary system. Access to thegastrointestinal tract, which can also rapidly introduce substances tothe blood stream, can be gained using oral enema, or injectable forms ofadministration. Compositions may be administered as a bolus injection orspray, or administered sequentially over time (episodically) such asevery two, four, six or eight hours, every day (QD) or every other day(QOD), or over longer periods of time such as weeks to months.Compositions may also be administered in a timed-release fashion such asby using slow-release resins and other timed or delayed releasematerials and devices.

Where systemic administration is desired, orally active compositions arepreferred as oral administration is a convenient and economical mode ofdrug delivery. Oral compositions may be poorly absorbed through thegastrointestinal lining. Compounds which are poorly absorbed tend to behighly polar. Preferably, such compositions are designed to reduce oreliminate their polarity. This can be accomplished by known means suchas formulating the oral composition with a complimentary reagent whichneutralizes its polarity, or by modifying the compound with aneutralizing chemical group. Preferably, the molecular structure issimilarly modified to withstand very low pH conditions and resist theenzymes of the gastric mucosa such as by neutralizing an ionic group, bycovalently bonding an ionic interaction, or by stabilizing or removing adisulfide bond or other relatively labile bond.

Treatments to the patient may be therapeutic or prophylactic.Therapeutic treatment involves administration of one or morecompositions of the invention to a patient suffering from one or moresymptoms of the disorder. Relief and even partial relief from one ormore symptoms can correspond to an increased life span or simply anincreased quality of life. Further, treatments that alleviate apathological symptom can allow for other treatments to be administered.

The term “compatible”, as used herein, means that the components of thecompositions are capable of being commingled with the CD1d blockingagents of the present invention, and with each other, in a manner suchthat does not substantially impair the desired efficacy.

Doses of the pharmaceutical compositions of the invention will varydepending on the subject and upon the particular route of administrationused. Dosages can range from 0.1 to 100,000 μg/kg per day, morepreferably 1 to 10,000 μg/kg. By way of an example only, an overall doserange of from about, for example, 1 microgram to about 300 microgramsmight be used for human use. This dose can be delivered at periodicintervals based upon the composition.

EXAMPLES

Animals

8-12 week old female C57B1/6 mice were purchased from Taconic Farms.Inc. (Germantown, N.Y.). The CD1d-null allele (generated in a129×C57B1/6 founder (25) (26) was backcrossed 7 generations into theC57B1/6 strain. (15) Homozygous CD1d deficient mice were obtained fromlittermate pairings and used to generate heterozygote CD1d deficient andwild type controls. Breeding pairs of mice deficient for CD1d on theBALB/c background were purchased from Jackson Labs (Bar Harbor, Me.).The C57B1/6 and BALB/c J 281 null mice were established by specificdeletion of the J 281 gene segment (27) (28). All mouse experiments wereapproved and conducted under IACUC guidelines.

Reagents

Contact hapten, 4-ethoxymethylene-2-phenyl-2-phenyl-2-oxazolin-5-one(oxazolone) was purchased from SIGMA, GalCer[(2S,3S,4R)-1-O-(-D-galactopyranosyl)-2-(N-hexacosanoylamino)-1,3,4-octadecanetriolwas kindly provided by Pharmaceutical Research Laboratories, KirinBrewery, Gunma, Japan, and CD1d-binding antagonists Polyethylene glycol(PEG)₂₀₀₀ dipalmitoyl-L-phosphatidylethanolamine (DPPE-PEG) and(PEG)₂₀₀₀ ceramide were purchased from Northern Lipids, Inc.

Contact Hypersensitivity

Mice at least seven weeks of age were sensitized epicutaneously on day 0with 70 μl of 4% 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (oxazolone,Sigma, St. Louis, Mo.) in acetone/olive oil (4/1) and challenged fivedays later on the right ear with 20 μl of 0.5% oxazolone or carrier onlyon the left ear. The hapten-specific increase in ear thickness at 24hours was determined with a micrometer. For experiments using DPPE-PEG,4% oxazolone in acetone/olive oil (4/1) solutions with or withoutDPPE-PEG (in dosages ranging from 5 mg/ml to 100 mg/ml) were applied.Finally, a mixture off 0.5% oxazolone and 50 mg/ml DPPE-PEG was used forthe inhibition of oxazalone recall.

Analysis of invariant Vα14Jα281 TCR frequency. Total RNA was isolatedfrom spleens of individual mice using TRIZOL (Gibro BRL, Grand Island,N.Y.) according to the manufacturer's recommendations. First-strand cDNAsynthesis was performed using oligo(dT) as a primer for reversetranscription of 2 μg of total RNA in a 50 μl reaction mixture usingMMLV-RT (Life Technologies, GIBCO-BRL, Gaithersgurg, Mass.).Quantitative analysis of Vα14Jα281 T cell frequency was done usingmultiplex RT-PCR by comparing the intensity of the TCR α-chain CDR3 bandwith the invariant INKT cell specific band, as previously described(29).

Treatment Protocols

Mice received a series of 5 intraperitoneal injections of either 2 μg ofαManCer (2 μg i.p. diluted in a solution of phosphate buffered salineand 0.5% Tween-20 every other day, starting 3 days prior to skinsensitization and continuing through to re-challenge.

Statistical Analysis

Significant differences between groups were evaluated with aMann-Whitney test, or where appropriate, a two tailed Student's t test.Mean differences were considered significant when p<0.05.

Contact hypersensitivity is ablated both in CD1d knock out and in iNKTcell-deficient Jα281 knock out mice. Mice were sensitized epicutaneouslyon day 0 with oxazolone and challenged six days later on the ear withoxazolone or carrier only, as described above. The hapten-specificincrease in ear thickness at 24 hours was determined with a micrometer.(See FIG. 1).

To examine the functional importance of CD1 d restricted iNKT cells inCHS we measured the ability of iNKT cell-deficient mice to generatecontact hypersensitivity to oxazolone. This reaction is a form ofdelayed-type hypersensitivity in which hapten-protein conjugates arepresented by cutaneous dendritic cells, followed by their migration toregional lymph nodes, to hapten-specific CD4 and CD8 positive Tlymphocytes (1)′(2)′(30)′(3). Upon secondary hapten challenge,sensitized T cells initiate a local inflammatory response. In thissystem, activation of iNKT cells by CD1d on skin DC is thought to be animportant initiation step followed by the elaboration of cytokines suchas IL-4 (10) (31). Interestingly, contact hypersensitivity to oxazolonewas significantly impaired in INKT cell-deficient mice on both theC57B1/6 and Balb/c backgrounds (FIG. 1), revealing a requirement forCD1d-restricted T cells for maximal hapten-specific immunity.

Systemical administration of an inactive CD1d binding lipid, α-ManCerinhibits contact hypersensitivity to oxazolone. Since CD1d- orJα281-null mice had impaired CSH responses, we examined whetheractivation or inhibition of iNKT cells with the protypic CD1d-dependentglycolipid agonist α-GalCer or antagonist α-ManCer, a glycolipid that isknown to bind CD1d but fails to activate iNKT cells, would modulate CSHresponses. Mice were treated with the α-GalCer or α-ManCer by i.p.injections administered prior to and during the sensitization phase withoxazolone. Pretreatment of mice with α-ManCer inhibited the CSH responseby 50% when compared to the control group (FIG. 2). Conversely,administration of α-GalCer did not alter the CSH response to oxazolone.Thus, CHS responses were not augmented by iNKT cell activation, butcould be significantly inhibited by systemic treatment with CD1dantagonists previously demonstrated not to activate iNKT cells in vivoand in vitro.

Topical DPPE-PEG (a CD1d-binding lipid) inhibits oxazolone-inducedcontact hypersensitivity (FIG. 3). Mice were sensitized epicutaneouslyon day 0 with oxazolone with or without DPPE-PEG (100 mg/ml) andchallenged five days later on the ear with oxazolone or carrier only, asdescribed above. The hapten-specific increase in ear thickness at 24hours was determined with a micrometer.

Topical administration of iNKT antagonist lipids inhibited thegeneration of CHS. Increasing doses of DPPE-PEG inhibitoxazolone-induced hypersensitivity in a dose-dependent manner, as shownin FIG. 4A. We evaluated whether administration of CD1d antagonistscould be used topically to block the generation of CSH responses. Sincea synthetically modified lipid, DPPE-PEG, was recently demonstrated tobe a significantly more soluble and effective competitor of α-GalCerpresentation than α-ManCer and was available in pharmacologicquantities, (22) this reagent was chosen for use as a topical inhibitorof CSH. In control experiments systemic administration of DPPE-PEG orPEG-ceramide inhibited CHS as effectively as α-ManCer (data not shown).For these experiments, wild type C57B1/6 mice were sensitized withoxazolone mice in the presence of increasing doses of DPPE-PEG.Half-maximal inhibition of CSH was achieved at 25 mg/ml of DPPE-PEG(FIG. 4A) and CSH could was inhibited in a dose response fashion.

FIG. 4B shows that topical DPPE-PEG inhibits contact hypersensitivityduring recall and priming. Inclusion of DPPE-PEG can inhibit recall tooxazolone even after priming with the agent. To determine whether theinhibitory effect of DPPE-PEG was dependent on when the inhibitor wasused, oxazolone-induced CSH responses were tested when DPPE-PEG waspresent at both phases, or only during priming or recall. Contacthypersensitivity responses were maximally inhibited by the inclusion ofDPPE-PEG during both the challenge and recall portions of the experiment(FIG. 4B). Notably, the CSH responses were also inhibited, but to alesser extent, when the reagent was used only during the challenge orrecall phases of the response. Importantly, the effect was specific forDPPE-PEG since neither palmitic acid, nor PEG alone were effectiveinhibitors of CSH (FIG. 2 and data not shown).

Inhibition of CSH by DPPE-PEG is strain independent (FIG. 5). The CD1dantigen presentation system can respond rapidly to cues in theenvironment and this function has been conserved since the evolutionarydivergence of rodents and humans (32) (24). To test whether the effectof DPPE-PEG was MHC or strain dependent, oxazalone-induced CSH wasdetermined for 4 additional MHC-unrelated congenic mouse strains in thepresence or absence of the inhibitor. The addition of DPPE-PEG was aneffective inhibitor in all strains tested (FIG. 5). There was, however,significant strain variability in both the magnitude of the responses tooxazolone and the ability of DPPE-PEG to inhibit CSH. Therefore,DPPE-PEG was an effective inhibitor of oxazolone-induced CSH in aMHC-independent fashion and in all strains tested.

CSH reactions were inhibited in mice deficient in iNKT cells directlyconfirming an important role for iNKT cells in CSH. Furthermore, CHS wasspecifically inhibited in those mice that were treated with CD1d-bindingantagonists. Thus, blockade of the CD1d system could be used to treatACD in an antigen- and MHC-independent fashion.

REFERENCES

-   1. Eisen, H. N., L. Orris, and S. Belman. 1952. Elicitation of    delayed allergic skin reactions with haptens: the dependence of    elicitation on hapten combination with protein. Journal of    Experimental Medicine 95:473.-   2. Silberberg, I., R. L. Baer, and S. A. Rosenthal. 1976. The role    of Langerhans cells in allergic contact hypersensitivity. A review    of findings in man and guinea pigs. J Invest Dermatol 66, no. 4:210.-   3. Gocinski, B. L., and R. E. Tigelaar. 1990. Roles of CD4+ and CD8+    T cells in murine contact sensitivity revealed by in vivo monoclonal    antibody depletion. J Immunol 144, no. 11:4121.-   4. Dieli, F., M. Taniguchi, G. L. Asherson, G. Sireci, N.    Caccamo, E. Scire, C. T. Bonanno, and A. Salerno. 1998. Development    of hapten-induced IL-4-producing CD4+ T lymphocytes requires early    IL-4 production by alphabeta T lymphocytes carrying invariant    V(alpha)14 TCR alpha chains. Int Immunol 10, no. 4:413.-   5. Ptak, W., and P. W. Askenase. 1992. Gamma delta T cells assist    alpha beta T cells in adoptive transfer of contact sensitivity. J    Immunol 149, no. 11:3503.-   6. Yokozeki, H., K. Watanabe, K. Igawa, Y. Miyazaki, I. Katayama,    and K. Nishioka. 2001. Gammadelta T cells assist alphabeta T cells    in the adoptive transfer of contact hypersensitivity to    para-phenylenediamine. Clin Exp Immunol 125, no. 3:351.-   7. Medzhitov, R., and C. Janeway, Jr. 2000. Innate immune    recognition: mechanisms and pathways. Immunol Rev 173:89.-   8. Watanabe, N., K. Ikuta, S. Fagarasan, S. Yazumi, T. Chiba, and T.    Honjo. 2000. Migration and differentiation of autoreactive B-1 cells    induced by activated gamma/delta T cells in antierythrocyte    immunoglobulin transgenic mice. J Exp Med 192, no. 11:1577.-   9. Ishii, N., K. Takahashi, H. Nakajima, S. Tanaka, and P. W.    Askenase. 1994. DNFB contact sensitivity (C S) in BALB/c and C3H/He    mice: requirement for early-occurring, early-acting,    antigen-specific, C S-initiating cells with an unusual phenotype    (Thy-1+, CD5+, CD3−, CD4−, CD8−, sIg−, B220+, MHC class II−, CD23+,    IL-2R−, IL-3R+, Mel-14−, Pgp-1+, J11d+, MAC-1+, LFA-1+, and Fc gamma    RII+). J Invest Dermatol 102, no. 3:321.-   10. Askenase, P. W. 2001. Yes T cells, but three different T cells    (alphabeta, gammadelta and N K T cells), and also B-1 cells mediate    contact sensitivity. Clin Exp Immunol 125, no. 3:345.-   11. Porcelli, S. A., and R. L. Modlin. 1999. The CD1 system:    antigen-presenting molecules for T cell recognition of lipids and    glycolipids. Annu Rev Immunol 17:297.-   12. Godfrey, D. I., K. J. Hammond, L. D. Poulton, M. J. Smyth,    and A. G. Baxter. 2000. NKT cells: facts, functions and fallacies.    Immunol Today 21, no. 11:573.-   13. Baxter, A. G., S. J. Kinder, K. J. L. Hammond, R. Scollay,    and D. I. Godfrey. 1997. Association between αβTCR+CD4−CD8− T-cell    deficiency and IDDM in NOD/Lt mice. Diabetes 46:572.-   14. Wilson, S. B., S. C. Kent, K. T. Patton, T. Orban, R. A.    Jackson, M. Exley, S. Porcelli, D. A. Schatz, M. A. Atkinson, S. P.    Balk, J. L. Strominger, and D. A. Hafler. 1998. Extreme Th1 bias of    invariant Valpha24JalphaQ T cells in type 1 diabetes. Nature 391,    no. 6663:177.-   15. Naumov, Y. N., K. S. Bahjat, R. Gausling, R. Abraham, M. A.    Exley, Y. Koezuka, S. B. Balk, J. L. Strominger, M. Clare-Salzer,    and S. B. Wilson. 2001. Activation of CD1d-restricted T cells    protects NOD mice from developing diabetes by regulating dendritic    cell subsets. Proc Natl Acad Sci USA 98, no. 24:13838.-   16. Cui, J., T. Shin, T. Kawano, H. Sato, E. Kondo, I. Toura, Y.    Kaneko, H. Koseki, M. Kanno, and M. Taniguchi. 1997. Requirement for    Vα14 NKT cells in IL-12-mediated rejection of tumors. Science    278:1623.-   17. Smyth, M. J., K. Y. T. Thia, S. E. A. Street, E. Cretney, J. A.    Trapani, M. Taniguchi, K. Tetsu, S. B. Pelikan, N. Y. Crowe,    and D. I. Godfrey. 2000. Differential Tumor Surveillance by Natural    Killer (N K) and NKT Cells. J. Exp. Med. 191, no. 4:661.-   18. Kitamura, H., K. Iwakabe, T. Yahata, S. Nishimura, A. Ohta, Y.    Ohmi, M. Sato, K. Takeda, K. Okumura, L. Van Kaer, T. Kawano, M.    Taniguchi, and T. Nishimura. 1999. The natural killer T (NKT) cell    ligand alpha-galactosylceramide demonstrates its immunopotentiating    effect by inducing interleukin (IL)-12 production by dendritic cells    and IL-12 receptor expression on NKT cells. J Exp Med 189, no.    7:1121.-   19. Tomura, M., W.-G. Yu, H.-J. Ahn, M. Yamashita, Y.-F. Yang, S.    Ono, T. Hamaoka, T. Kawano, M. Taniguchi, Y. Koezuka, and H.    Fujiwara. 1999. A Novel Function of Valpha14+CD4+NKT Cells:    Stimulation of IL-12 Production by Antigen-Presenting Cells in the    Innate Immune System. J. Immunol 163:93.-   20. Toura, I., T. Kawano, Y. Akutsu, T. Nakayama, T. Ochiai, and M.    Taniguchi. 1999. Cutting edge: inhibition of experimental tumor    metastasis by dendritic cells pulsed with alpha-galactosylceramide.    J Immunol 163, no. 5:2387.-   21. Zeng, Z.-H., A. R. Castano, B. W. Segelke, E. A. Stura, P. A.    Peterson, and I. A. Wilson. 1997. Crystal structure of mouse CD1:an    MHC-like fold with a large hydrophobic binding groove. Science 277,    no. 5324:339.-   22. Naidenko, O. V., J. K. Maher, W. A. Ernst, T. Sakai, R. L.    Modllin, and M. Kronenberg. 1999. Binding and Antigen Presentation    of Ceramide-containing glycolipids by Soluble Mouse and Human CD1d    Molecules. J. Exp. Med. 190, no. 8:1069.-   23. Joyce, S., A. S. Woods, J. W. Yewdell, J. R. Bennink, A. D. De    Silva, A. Boesteanu, S. P. Balk, R. J. Cotter, and R. R.    Brutkiewicz. 1998. Natural ligand of mouse CD1d1: cellular    glycosylphosphatidylinositol. Science 279, no. 5356:1541.-   24. Brossay, L., and M. Kronenberg. 1999. Highly conserved    antigen-presenting function of CD1d molecules. Immunogenetics 50,    no. 3-4:146.-   25. Sonoda, K. H., M. Exley, S. Snapper, S. P. Balk, and J.    Stein-Streilein. 1999. CD1-reactive natural killer T cells are    required for development of systemic tolerance through an    immune-privileged site [see comments]. J Exp Med 190, no. 9:1215.-   26. Exley, M. A., N. J. Bigley, O. Cheng, S. M. Tahir, S. T.    Smiley, Q. L. Carter, H. F. Stills, M. J. Grusby, Y. Koezuka, M.    Taniguchi, and S. P. Balk. 2001. CD1d-reactive T-cell activation    leads to amelioration of disease caused by diabetogenic    encephalomyocarditis virus. J Leukoc Biol 69, no. 5:713.-   27. Smiley, S. T., M. H. Kaplan, and M. J. Grusby. 1997.    Immunoglobulin E production in the absence of    interleukin-4-secreting CD1-dependent cells. Science 275:977.-   28. Kawano, T., J. Cui, Y. Koezuka, I. Toura, Y. Kaneko, K.    Motoki, H. Ueno, R. Nakagawa, H. Sato, E. Kondo, H. Koseki, and M.    Taniguchi. 1997. CD1d-restricted and TCR-mediated activation of    valpha14 NKT cells by glycoceramides. Science 278:1626.-   29. Gorski, J., M. Yassai, X. Zhu, B. Kissela, B. Kissella, C.    Keever, and N. Flomenberg. 1994. Circulating T cell repertoire    complexity in normal individuals and bone marrow recipients analyzed    by CDR3 size spectratyping. Correlation with immune status. J    Immunol 152, no. 10:5109.-   30. Bacci, S., P. Alard, R. Dai, T. Nakamura, and J. W.    Streilein. 1997. High and low doses of haptens dictate whether    dermal or epidermal antigen-presenting cells promote contact    hypersensitivity. Eur J Immunol 27, no. 2:442.-   31. Gerlini, G., H. P. Hefti, M. Kleinhans, B. J. Nickoloff, G.    Burg, and F. O. Nestle. 2001. Cd1d is expressed on dermal dendritic    cells and monocyte-derived dendritic cells. J Invest Dermatol 117,    no. 3:576.-   32. Bendelac, A., M. N. Rivera, H.-S. Park, and J. H. Roark. 1997.    Mouse CD1-Specific NK1 T Cells: Development, Specificity, and    Function. Annual Review of Immunology 15:535.-   33. Hopkin, J. M. 1997. Mechanisms of enhanced prevalence of asthma    and atopy in developed countries. Curr Opin Immunol 9, no. 6:788.-   34. Akdis, C. A., M. Akdis, A. Trautmann, and K. Blaser. 2000.    Immune regulation in atopic dermatitis. Curr Opin Immunol 12, no.    6:641.-   35. Girolomoni, G., S. Sebastiani, C. Albanesi, and A. Cavani. 2001.    T-cell subpopulations in the development of atopic and contact    allergy. Curr Opin Immunol 13, no. 6:733.-   36. Apostolou, I., Y. Takahama, C. Belmant, T. Kawano, M. Huerre, G.    Marchal, J. Cui, M. Taniguchi, H. Nakauchi, J. J. Fournie, P.    Kourilsky, and G. Gachelin. 1999. Murine natural killer T(NKT) cells    [correction of natural killer cells] contribute to the granulomatous    reaction caused by mycobacterial cell walls. Proc Natl Acad Sci USA    96, no. 9:5141.-   37. Mempel, M., B. Flageul, F. Suarez, C. Ronet, L. Dubertret, P.    Kourilsky, G. Gachelin, and P. Musette. 2000. Comparison of the T    cell patterns in leprous and cutaneous sarcoid granulomas. Presence    of Valpha24-invariant natural killer T cells in T-cell-reactive    leprosy together with a highly biased T cell receptor Valpha    repertoire. Am J Pathol 157, no. 2:509.-   38. Mempel, M., C. Ronet, F. Suarez, M. Gilleron, G. Puzo, L. Van    Kaer, A. Lehuen, P. Kourilsky, and G. Gachelin. 2002. Natural Killer    T Cells Restricted by the Monomorphic MHC Class Ib CD1d1 Molecules    Behave Like Inflammatory Cells. Journal of mnmunology 168:365.-   39. Sebastiani, S., P. Allavena, C. Albanesi, F. Nasorri, G.    Bianchi, C. Traidl, S. Sozzani, G. Girolomoni, and A. Cavani. 2001.    Chemokine receptor expression and function in CD4+ T lymphocytes    with regulatory activity. J Immunol 166, no. 2:996.-   40. Cavani, A., F. Nasorri, C. Prezzi, S. Sebastiani, C. Albanesi,    and G. Girolomoni. 2000. Human CD4+ T lymphocytes with remarkable    regulatory functions on dendritic cells and nickel-specific Th1    immune responses. J Invest Dermatol 114, no. 2:295.-   41. Bendelac, A., R. D. Hunziker, and O. Lantz. 1996. Increased    interleukin 4 and immunogloulin E production in transgenic mice    overexpressing NK1 T cells. Journal of Experimental Medicine    184:1285.-   42. Mendiratta, S. K., W. D. Martin, S. Hong, A. Boesteanu, S.    Joyce, and L. Van Kaer. 1997. CD1d1 mutant mice are deficient in    natural T cells that promptly produce IL-4. Immunity 6:469.-   43. Wilson, S. B., S. C. Kent, H. F. Horton, A. A. Hill, P. L.    Bollyky, D. A. Hafler, J. L. Strominger, and M. C. Byrne. 2000.    Multiple differences in gene expression in regulatory    Valpha24JalphaQ T cells from identical twins discordant for type I    diabetes. Proc Natl Acad Sci USA 97, no. 13:7411.-   44. Yang, O. O., F. K. Racke, P. T. Nguyen, R. Gausling, M. E.    Severino, H. F. Horton, M. C. Byrne, J. L. Strominger, and S. B.    Wilson. 2000. CD1d on Myeloid Dendritic Cells Stimulates Cytokine    Secretion and Cytolytic Activity of Valpha24JalphaQ T Cells: A    Feedback Mechanism for Immune Regulation. J. Immunol. 165, no.    7:3756.-   45. Exley, M., J. Garcia, S. P. Balk, and S. Porcelli. 1997.    Requirements for CD1d Recognition by Human Invariant Vα24+CD4−CD8− T    Cells. The Journal of Experimental Medicine 186:1.-   46. Kadowaki, N., S. Antonenko, S. Ho, M. C. Rissoan, V.    Soumelis, S. A. Porcelli, L. L. Lanier, and Y. J. Liu. 2001.    Distinct cytokine profiles of neonatal natural killer t cells after    expansion with subsets of dendritic cells. J Exp Med 193, no.    10:1221.-   47. Ikarashi, Y., R. Mikami, A. Bendelac, M. Terme, N. Chaput, M.    Terada, T. Turz, E. Angevin, F. A. Lemonnier, H. Wakasugi, and L.    Zitvogel. 2001. Dendritic Cell Maturation Overrules H-2D-mediated    Natural Killer T (NKT) Cell Inhibition: Critical Role for B7 in    CD1d-dependent NKT Cell Interferon gamma Production. Journal of    Experimental Medicine 194, no. 8:1179.-   48. Rissoan, M. C., V. Soumelis, N. Kadowaki, G. Grouard, F.    Briere, R. de Waal Malefyt, and Y. J. Liu. 1999. Reciprocal control    of T helper cell and dendritic cell differentiation [see comments].    Science 283, no. 5405:1183.-   49. Shreedhar, V., A. M. Moodycliffe, S. E. Ullrich, C.    Bucana, M. L. Kripke, and L. Flores-Romo. 1999. Dendritic cells    require T cells for functional maturation in vivo. Immunity 11, no.    5:625.

All references described herein are incorporated herein by reference.

What is claimed is:
 1. A composition formulated for topicaladministration comprising: (a) an agent that attenuates CD1d-restrictedNK T cell responses, wherein 1) the agent is a phospholipid conjugatedto polyethylene glycol (PEG) that binds CD1d and i) inhibits activationof CD1d-restricted NK T cells or ii) blocks CD1d-specific receptors, 2)the phospholipid is 1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine(DPPE), and 3) the DPPE is present in an amount greater than 2.5% weightper volume; and (b) a carrier for topical administration, wherein thecarrier is a vegetable oil.
 2. The composition of claim 1, wherein theagent is present in an amount between 2.5% weight per volume and 10.0%weight per volume.
 3. The composition of claim 1, wherein the agent is1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-[Poly (ethyleneglycol) 2000] (DPPE-PEG).